Mutant GGGGCC RNA prevents YY1 from binding to Fuzzy promoter which stimulates Wnt/β-catenin pathway in C9ALS/FTD

The GGGGCC hexanucleotide repeat expansion mutation in the chromosome 9 open reading frame 72 (C9orf72) gene is a major genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia (C9ALS/FTD). In this study, we demonstrate that the zinc finger (ZF) transcriptional regulator Yin Yang 1 (YY1) binds to the promoter region of the planar cell polarity gene Fuzzy to regulate its transcription. We show that YY1 interacts with GGGGCC repeat RNA via its ZF and that this interaction compromises the binding of YY1 to the FuzzyYY1 promoter sites, resulting in the downregulation of Fuzzy transcription. The decrease in Fuzzy protein expression in turn activates the canonical Wnt/β-catenin pathway and induces synaptic deficits in C9ALS/FTD neurons. Our findings demonstrate a C9orf72 GGGGCC RNA-initiated perturbation of YY1–Fuzzy transcriptional control that implicates aberrant Wnt/β-catenin signalling in C9ALS/FTD-associated neurodegeneration. This pathogenic cascade provides a potential new target for disease-modifying therapy.

The GGGGCC hexanucleotide repeat expansion mutation in the chromosome 9 open reading frame 72 (C9orf72) gene is a major genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia (C9ALS/FTD).In this study, we demonstrate that the zinc finger (ZF) transcriptional regulator Yin Yang 1 (YY1) binds to the promoter region of the planar cell polarity gene Fuzzy to regulate its transcription.We show that YY1 interacts with GGGGCC repeat RNA via its ZF and that this interaction compromises the binding of YY1 to the Fuzzy YY1 promoter sites, resulting in the downregulation of Fuzzy transcription.The decrease in Fuzzy protein expression in turn activates the canonical Wnt/ β-catenin pathway and induces synaptic deficits in C9ALS/FTD neurons.Our findings demonstrate a C9orf72 GGGGCC RNA-initiated perturbation of YY1-Fuzzy transcriptional control that implicates aberrant Wnt/β-catenin signalling in C9ALS/FTD-associated neurodegeneration.This pathogenic cascade provides a potential new target for disease-modifying therapy.
The non-coding hexanucleotide GGGGCC repeat expansion mutation in the chromosome 9 open reading frame 72 (C9orf72) gene is a major genetic cause of amyotrophic lateral sclerosis (ALS), a progressive and fatal neurodegenerative disorder characterised by a loss of motor neurons and paralysis 1,2 .Frontotemporal dementia (FTD), in which the loss of prefrontal cortical neurons and their connections leads to behavioural change, shares genetic and pathological features with ALS, exemplified by the fact that mutation in the C9orf72 gene underlies a significant proportion of FTD cases 3 .C9ALS/FTD has been used to collectively refer to patients with clinical features of ALS and/or FTD.Three non-exclusive pathogenic mechanisms have been implicated in C9ALS/FTD: (1) RNA-mediated toxicity 4 ; (2) repeat-associated non-ATG translation of dipeptide proteins 5 produced by both sense and antisense strands of the pathogenic allele, which induce neuronal dysfunction by interacting with key cellular proteins; and (3) loss of endogenous C9orf72 protein function due to reduced C9orf72 transcription resulting from the GGGGCC repeat expansion (C9orf72 haploinsufficiency) 6 .Elucidating the effects of GGGGCC repeat expansion on molecular pathways is essential for a better understanding of the disease mechanisms underlying C9ALS/FTD.
Yin Yang 1 (YY1) is a versatile transcriptional regulator that ensures proper transcriptional profiling in neurons 7 .In individuals with neurodegenerative disorders, the function of YY1 is compromised as it is recruited to disease protein aggregates 8 , mislocalised within the cell 9 and/or proteolytically degraded 10,11 .Currently, the role of YY1 in C9ALS/FTD pathogenesis is unclear.Yin et al. 9 reported that when compared with the astrocytes isolated from a mouse model overexpressing the wild-type form of human superoxide dismutase 1 (SOD1), the concentration of nuclear YY1 protein was increased in the astrocytes carrying an ALS-causing SOD1 G93A mutation.The accumulation of nuclear YY1 attenuates the expression of excitatory amino acid transporter-2, a protein that mediates glutamate uptake from the extracellular compartment 12 .This attenuation in turn leads to insufficient glutamate clearance and neuronal excitotoxicity 9 .
The Fuzzy protein is classified as one of the ciliogenesis and planar polarity effector (CPLANE) proteins, which comprise additional members, including Inturned, Wdpcp, Rsg1 and Jbts17 13 .Different CPLANE members control protein transport into, along and out of ciliary axonemes in support of ciliary outgrowth in vertebrate cells 13 .Cell migration defects that lead to developmental neural tube closure defects have been observed in Fuzzy-null (Fuzzy −/− ) mutant mice 14 .In humans, mutations in Fuzzy are known to cause neural tube defects 15 .Recently, Fuzzy has been implicated in human neurodegenerative disorders 8 and cancer [16][17][18] .We previously reported that YY1 is recruited to polyglutamine aggregates and amyloid beta peptide 42 (Aβ 1-42 ), which reduces the binding of YY1 to the Fuzzy gene promoter and upregulates the expression of the Fuzzy protein 8 .An increase in Fuzzy expression triggers the activation of a mitogen-activated protein kinase (MAPK)-caspase pro-apoptotic signalling cascade and results in neuronal cell death 8 .
Wnt signalling controls numerous essential cellular behaviours such as cell proliferation, polarity and mobility 19 .Depending on the nuclear factors involved, Wnt signalling can be categorised into three major pathways: the canonical Wnt/β-catenin pathway and the noncanonical Wnt/PCP and Wnt/Ca 2+ pathways 20 .Several lines of evidence highlight the role of Wnt signalling in human neurodegenerative disorders.For example, in Alzheimer's disease models, activating the Wnt/β-catenin pathway ameliorates neurodegeneration by inhibiting Aβ 1-42 production and tau protein hyperphosphorylation 21 .Interestingly, tight regulation of the Wnt pathway by Fuzzy has been shown to be essential for embryonic maturation.Embryonic fibroblasts isolated from Fuzzy −/− mice demonstrated hyperactivation of the canonical Wnt/β-catenin pathway 22 .The expression of Wnt/β-catenin-targeted genes was reported to be upregulated in Fuzzy −/− mouse embryos 23 .However, the relationship between Fuzzy and Wnt signalling remains to be established in the context of human neurodegenerative disorders.
In this study, we demonstrated that Fuzzy gene transcription is downregulated in C9ALS/FTD-induced pluripotent stem cell (iPSC)derived spinal motor neurons.Mechanistically, we demonstrated that mutant GGGGCC RNA, and not dipeptide repeat (DPR) proteins or C9orf72 haploinsufficiency, leads to the downregulation of Fuzzy expression.We further showed that this downregulation involves two YY1-binding sites (Fuzzy YY1-a and Fuzzy YY1-b ) in the Fuzzy promoter.Mutant GGGGCC RNA interacts with YY1 and prevents it from binding to both Fuzzy YY1-a and Fuzzy YY1-b sites.The reduction in Fuzzy protein expression induces the expression of the Wnt/β-catenin-responsive gene Paired-like Homeodomain 2 (PITX2).We further showed that silencing PITX2 expression rescues cell death and synaptic defects in our C9ALS/FTD models.This work provides the link between Wnt/βcatenin pathway activation and C9ALS/FTD pathogenicity.Taken together, our data revealed a C9ALS/FTD pathogenic pathway initiated by mutant GGGGCC RNA, which induces synaptic deficits and neuronal cell death.We also showed that targeting this pathway ameliorates C9ALS/FTD-associated neurodegeneration.

Fuzzy expression is downregulated in C9ALS/FTD spinal motor neurons
We differentiated both control and C9ALS/FTD iPSCs into spinal motor neurons (Supplementary Fig. 1a 24 ), and examined the transcript levels of CPLANE genes 13 , Fuzzy, Inturned, Wdpcp, Rsg1 and Jbts17, in four independently derived patient neurons.When compared with the healthy control iPSC-derived spinal motor neurons, Fuzzy is the only CPLANE gene whose expression was significantly reduced in C9ALS/ FTD iPSC-derived spinal motor neurons (Fig. 1a; Supplementary Fig. 1b-e).A similar reduction was further detected in a pair of patient neurons and their GGGGCC repeat expansion-corrected isogenic control neuron (Fig. 1a 25 ).We also showed that Fuzzy protein level was reduced in C9ALS/FTD iPSC-derived spinal motor neurons (Fig. 1b).In contrast to the C9ALS/FTD samples, spinal motor neurons derived from patient iPSCs carrying TARDBP mutations 24 showed no alteration in Fuzzy expression (Supplementary Fig. 1f).These results suggest that Fuzzy downregulation is linked to the GGGGCC repeat expansion mutation in C9ALS/FTD spinal motor neurons.
Upon the deletion of the Fuzzy −1332/−1143 region, the mutant GGGGCC RNA's effect on Fuzzy promoter activity was diminished (Fig. 1f).Based on an in silico prediction, a putative transcriptional factor binding site, Fuzzy YY1-a (Supplementary Fig. 2e), was identified.We took a site-directed mutagenesis approach to examine the involvement of Fuzzy YY1-a in the mutant GGGGCC RNA-mediated downregulation of Fuzzy promoter activity.When a single nucleotide substitution, T to C, was introduced to the Fuzzy YY1-a site (Fig. 1g 28 ), the (GGGGCC) 66 -mediated downregulation of Fuzzy promoter activity was abolished (Fig. 1h).We previously identified another YY1 binding site, Fuzzy YY1-b , in the Fuzzy promoter region (Fig. 1g 8 ).In contrast to Fuzzy YY1-a , when the same T to C substitution was introduced to Fuzzy YY1-b site (Fig. 1g), no restoration of Fuzzy promoter activity in (GGGGCC) 66 -expressing cells was observed (Fig. 1i).Our data suggest that Fuzzy YY1-a is critical for the Fuzzy downregulation in mutant GGGGCC RNA-expressing cells.
YY1 binds to and plays opposing roles at Fuzzy YY1-a and Fuzzy YY1-b sites We carried out cell-free electrophoretic mobility shift assay (EMSA) to examine the binding affinities between YY1 protein and Fuzzy YY1 DNAs.We found that recombinant YY1 protein formed protein-DNA complexes with both Fuzzy YY1-a and Fuzzy YY1-b DNA probes (Fig. 2a).The AAV P5 DNA probe was used as a YY1-binding positive control (Supplementary Fig. 3a 29 ).The binding affinity of YY1-Fuzzy YY1-a (K d = 242.40± 21.66 nM) was ~1.6-fold higher than that of YY1-Fuzzy YY1-b (K d = 404.90± 45.85 nM), indicating that YY1 binds more strongly to Fuzzy YY1-a than to Fuzzy YY1-b (Fig. 2a).
Using a cell-based chromatin immunoprecipitation (ChIP) assay, we further demonstrated the binding of YY1 to both endogenous Fuzzy YY1-a and Fuzzy YY1-b promoter sites in SK-N-MC cells (Fig. 2b; Supplementary Fig. 3b, c).We also found that YY1 binds to the endogenous Fuzzy DNA sequence via its C-terminal zinc finger (ZF) domain (Fig. 2b; Supplementary Fig. 3b-d).The YY1 protein ZF domain carries four highly conserved ZF motifs (ZF1-4; Supplementary Fig. 3b, d 30,31    Fuzzy YY1-a and Fuzzy YY1-b .It was found that both full-length YY1 and YY1 ZF1+ZF2+ZF3 mutant proteins showed comparable binding capacities towards Fuzzy YY1-a .This indicates that ZF4 is not necessary for mediating YY1-Fuzzy YY1-a interaction.In contrast, all mutant YY1 proteins that lack ZF3, including YY1 ZF1+ZF2 , YY1 ZF1 and YY1 ΔZF , showed reduced Fuzzy YY1-a binding (Fig. 2b).This shows that the ZF3 motif of YY1 is a key determinant of Fuzzy YY1-a binding.Using the same approach, we found that the interaction between YY1 and Fuzzy YY1-b is mediated through ZF4 (Fig. 2b).These results highlight the distinct roles of ZF motifs in facilitating YY1-Fuzzy YY1-a and YY1-Fuzzy YY1-b interactions.YY1 controls gene transcription upon binding to gene promoters 32 .We studied the regulatory function of YY1 on Fuzzy transcription by introducing a known YY1-DNA binding disruption mutation 28 to Fuzzy YY1 sites (Fig. 1g).Inactivation of the Fuzzy YY1-a site led to a dramatic ~50-fold reduction in Fuzzy promoter activity in SK-N-MC cells (Fig. 2c).In contrast, inactivating Fuzzy YY1-b site only mildly potentiated Fuzzy promoter activity by ~2.5 fold (Fig. 2c).Our data clearly demonstrate that YY1 interacts with the Fuzzy YY1-a site to promote transcription activation, while it serves as a transcriptional repressor at the Fuzzy YY1-b site.
Reducing the binding of YY1 to Fuzzy YY1 promoter sites limits Fuzzy promoter activity We showed that mutant GGGGCC RNA downregulates Fuzzy transcription (Fig. 1a).Similarly, knockdown of YY1 (Supplementary Fig. 3e) caused inhibition of wild-type Fuzzy promoter activity (Fig. 2d).We next examined whether GGGGCC RNA would disrupt YY1-Fuzzy YY1 interaction, which consequently leads to the reduction of Fuzzy transcripts in patient neurons (Fig. 1a).To test this, we first performed EMSA to examine whether GGGGCC RNA perturbs the YY1-Fuzzy YY1 binding.Upon the addition of GGGGCC RNA, the binding of YY1 to both Fuzzy YY1-a and Fuzzy YY1-b sites were disrupted (Fig. 2e).When compared with the YY1-Fuzzy YY1-a complex formation, it is evident that the YY1-Fuzzy YY1-b complexes were more susceptible to GGGGCC RNA disturbance (Fig. 2e).We next performed cell-based ChIP assay to validate our cell-free EMSA findings, and found that the binding of YY1 to both Fuzzy YY1 sites was disrupted in the mutant (GGGGCC) 66 RNAexpressing cells (Fig. 2f; Supplementary Fig. 3f).Under the same mutant (GGGGCC) 66 expression condition, the YY1-Fuzzy YY1-a interaction showed a higher tolerance toward mutant GGGGCC RNA than YY1-Fuzzy YY1-b interaction (Fig. 2f).This is in line with our other observation (Fig. 2a) that YY1 has a higher binding affinity for Fuzzy YY1-a than for Fuzzy YY1-b .In summary, our findings support the notion that when compared to Fuzzy YY1-a , the binding of YY1 to Fuzzy YY1-b is more susceptible to mutant GGGGCC RNA disturbance (Fig. 2e, f).
We also examined the consequence of mutant GGGGCC RNAmediated disruption of YY1 binding on Fuzzy YY1 sites.We observed that a stepwise increase in mutant GGGGCC RNA expression caused a progressive disruption of YY1 binding to both Fuzzy YY1-a and Fuzzy YY1-b sites (Fig. 2f).Interestingly, such upward mutant GGGGCC RNA gradient also led to a gradual inhibition of Fuzzy promoter activity in our SK-N-MC cell model (Fig. 2g).In Fig. 2c, we showed a dramatic ~50fold reduction of Fuzzy promoter activity when the YY1-Fuzzy YY1-a interaction was perturbed.On the contrary, only a mild induction (~2.5fold) of Fuzzy activity was detected when the YY1-Fuzzy YY1-b interaction was abolished (Fig. 2c).Collectively, our data suggest that the mutant GGGGCC RNA-mediated downregulation of Fuzzy transcription is mainly attributable to the impairment of YY1-Fuzzy YY1-a binding, which translates into a pronounced inhibition of Fuzzy promoter activity.The mild induction effect mediated through the Fuzzy YY1-b site would likely be overshadowed by the Fuzzy YY1-a site.

GGGGCC RNA interacts with and sequesters YY1 to RNA foci
Other than interacting with DNAs, the YY1 protein also binds RNAs 33 .We continued to investigate whether GGGGCC RNA would interact with YY1.Our EMSA analysis showed that recombinant YY1 full-length protein bound directly to GGGGCC RNA at K d = 46.76± 3.42 nM (Fig. 3a), but less tightly to a randomised GC-rich RNA probe (K d = 200.60 ± 17.92 nM; Supplementary Fig. 4a) that carries equivalent number of G and C nucleotides as with our GGGGCC RNA probe (Supplementary Table 4).Similar to EMSA, the fluorescence polarisation (FP) assay has been widely used to study protein-nucleic acid interactions 34 .Consistent with EMSA, our FP assay results also demonstrate a direct binding between recombinant YY1 full-length protein and GGGGCC RNA (K d = 45.30± 3.67 nM; Fig. 3b).Notably, the binding affinity of YY1-GGGGCC RNA (K d = 46.76± 3.42 nM) was much stronger than that of YY1-Fuzzy YY1-a (K d = 242.40± 21.66 nM) and YY1-Fuzzy YY1-b (K d = 404.90± 45.85 nM) (Fig. 2a).Our data therefore support the notion that GGGGCC RNA interacts with YY1 and prevents it from binding to Fuzzy YY1 sites (Fig. 2e, f).This consequently leads to the downregulation of Fuzzy transcription (Fig. 2g).The PP7 RNA was used as a YY1-binding positive control (Supplementary Fig. 4b 33 ).We also detected a lower binding affinity between YY1 full-length protein and GGGGCC DNA (Supplementary Fig. 4c).
The expression of mutant GGGGCC RNA leads to the formation of RNA foci in SK-N-MC cells (Fig. 3c 35 ).Using in situ hybridisation followed by immunofluorescence, we showed that GGGGCC RNA foci colocalised with endogenous heterogeneous nuclear ribonucleoprotein H1 (hnRNP H), a known GGGGCC RNA-interacting protein (Fig. 3c; Supplementary Fig. 5a 36 ).Such a pattern of co-localisation was detected in ~80% of GGGGCC RNA foci-positive cells (Fig. 3f).Under the same experimental conditions, the co-localisation between endogenous YY1 and GGGGCC RNA foci was detected in ~40% of GGGGCC RNA foci-positive cells (Fig. 3d, f; Supplementary Fig. 5b).The subcellular co-localisation of YY1 protein with GGGGCC RNA foci was further confirmed using a separate anti-YY1 antibody that recognises a different epitope (Fig. 3e, f; Supplementary Fig. 5c).It is of note that the protein-foci co-localisation pattern was abolished upon treatment with RNase (Fig. 3c-e).This indicates that YY1 protein mis-localisation is RNA-mediated.The Fuzzy/Fuzzy transcript (a) and protein (b) levels were downregulated in C9ALS/FTD patient iPSC-derived spinal motor neurons when compared to the healthy and isogenic control neurons.c Illustration of the Fuzzy promoter luciferase reporter and different GGGGCC expression constructs used in this study.d Overexpression of pAG3-(GGGGCC) 66 construct suppressed Fuzzy promoter luciferase activities at Fuzzy -2732/+574 , Fuzzy -2032/+574 and Fuzzy -1332/+574 regions, whereas the activity of Fuzzy -632/+574 luciferase construct was not altered.The (GGGGCC) 66 -responsive region, Fuzzy -1332/-633 , is in yellow.e Fuzzy promoter activity was downregulated in (GGGGCC) 106 -RO-expressing cells, while such downregulation was restored upon treatment of BIND peptide.f The (GGGGCC) 66 -and (GGGGCC) 106 -RO-responsive element was further narrowed down to Fuzzy -1332/-1143 region, which is in red.g Schematic representation of the locations and sequences of Fuzzy YY1-a (in red font) and Fuzzy YY1-b (in blue font) sites in Fuzzy -1332/+574 promoter region."+1" represents the transcriptional initiation (arrow) site.The Fuzzy YY1-a and Fuzzy YY1-b sites were mutated upon introducing a single nucleotide substitution from "T" to "C" (in green font).h, i Mutation of Fuzzy YY1-a (h), but not Fuzzy YY1-b (i) abolished the (GGGGCC) 66 -mediated downregulation of Fuzzy -1332/+574 promoter activity.One-way ANOVA followed by post hoc Tukey's test was used for the comparisons between disease and healthy control neurons in panels (a) and (b).Oneway ANOVA followed by post hoc Dunnett's test was used for the comparisons between disease and isogenic control neurons in panels (a) and (b).Two-tailed unpaired Student's t-test was used in panels (d-f), (h) and (i).The exact P values are listed in Supplementary Table 7.For panels (a) and (b), n = 3 biologically independent experiments.For the rest of the panels, n = 5 biologically independent experiments.Data is presented as mean ± SEM.The illustrations in panels (c) and (g) were created using Adobe Illustrator and BioRender.com,respectively.Source data are provided as a Source Data file.
https://doi.org/10.1038/s41467-023-44215-wNature Communications | (2023) 14:8420 We further tested whether the co-localisation between YY1 and GGGGCC RNA foci decreased the mobility of YY1.Twelve copies of MS2 aptamers, which serve as binding sites for MS2-coat binding protein (MS2CP) 37 , were inserted downstream of the pAG3-(GGGGCC) 2/66 construct (Fig. 1c).Live cell imaging was performed on cells co-transfected with pAG3-(GGGGCC) 66 -MS2 and MS2CP-YFP constructs.The GGGGCC RNA foci were found to be co-localised with the fluorescently tagged YY1 protein (YY1-mCherry; Fig. 3g).We next performed fluorescence recovery after photobleaching assay on these cells and found a lack of fluorescent recovery of the YY1-mCherry protein upon photobleaching in GGGGCC RNA foci-positive cells (Fig. 3g).Similar findings were observed for another GGGGCC foci-recruited protein hnRNP H (Fig. 3h 36 ).As a control, we demonstrated that the co-transfection of pAG3-(GGGGCC) 2 -MS2 unexpanded control and MS2CP-YFP did not result in any detectable RNA foci formation (Fig. 3g).These results show that the intracellular mobility of YY1 was compromised in mutant GGGGCC RNA cells, presumably due to its association with the GGGGCC RNA and/or sequestration to the RNA foci.

YY1 ZF3 is involved in mediating YY1-GGGGCC RNA interaction
Zinc finger motifs of YY1 have been reported to mediate protein-RNA interactions 33 .We performed cell-free EMSA using two versions of recombinant mutant YY1 proteins, one carries ZF1-3 motifs (YY1 ZF1+ZF2+ZF3 ) and another one with only ZF1 and 2 (YY1 ZF1+ZF2 ) (Supplementary Fig. 6a), to determine the role of ZF motifs in YY1-GGGGCC RNA binding.When compared with YY1 full-length protein (K d = 46.76± 3.42 nM; Fig. 3a), YY1 ZF1+ZF2+ZF3 interacted with (GGGGCC) 8 RNA at a similar binding affinity (K d = 59.01 ± 1.23 nM; Fig. 4a).This suggests that the ZF4 motif is not crucial for GGGGCC RNA interaction.In contrast, the binding affinity between YY1 ZF1+ZF2 and (GGGGCC) 8 RNA was reduced to 129.43 ± 26.23 nM (Fig. 4a).Our data thus highlight that  4. b The ZF3 in YY1 is required for interacting with Fuzzy YY1-a , while ZF4 mediates the interaction between YY1 and Fuzzy YY1-b .Both Fuzzy YY1-a and Fuzzy YY1-b DNAs were amplified from the endogenous Fuzzy gene promoter.c Mutation of Fuzzy YY1-a led to downregulation of Fuzzy promoter activity, whereas mutation of Fuzzy YY1-b caused the opposite upregulation effect.d Knockdown of YY1 led to the inhibition of Fuzzy promoter activity.e Addition of GGGGCC RNA perturbed the formation of protein-DNA complexes between recombinant YY1 protein and Fuzzy YY1-a /Fuzzy YY1-b DNA, leading to the increase in levels of free Fuzzy YY1-a and Fuzzy YY1-b DNAs.The YY1-Fuzzy YY1-b binding is more susceptible to GGGGCC RNA interference.f The binding of endogenous YY1 protein to Fuzzy YY1-a and Fuzzy YY1-b sites was impaired upon overexpression of the pAG3-(GGGGCC) 66 construct.The YY1-Fuzzy YY1-b interaction was more prone to be disturbed in the presence of mutant GGGGCC RNA.Both Fuzzy YY1-a and Fuzzy YY1-b DNAs were amplified from the endogenous Fuzzy promoter.g The increase of the transfection amount of pAG3-(GGGGCC) 66 plasmid gradually inhibited Fuzzy promoter activity.One-way ANOVA followed by post hoc Tukey's test and one-way ANOVA followed by post hoc Dunnett's test were used in panels (b) and (c), respectively.Two-tailed unpaired Student's t-test was used in panels (d), (f) and (g).The exact P values are listed in Supplementary Table 7.For panels (c), (d) and (g), n = 5 biologically independent experiments.For the rest of the panels, n = 3 biologically independent experiments.Data is presented as mean ± SEM.Source data are provided as a Source Data file.7. n = 3 biologically independent experiments and data is presented as mean ± SEM.Source data are provided as a Source Data file.
the YY1 ZF3 motif is important for mediating YY1 and GGGGCC RNA interaction.We further found that the ZF3 motif is one determinant for YY1 to co-localise with RNA foci (Fig. 4b, c; Supplementary Fig. 6b).

YY1 overexpression ameliorates cell death and synaptic defects in C9ALS/FTD models
The GGGGCC RNA foci were detected in C9ALS/FTD iPSC-derived spinal motor neurons, but not in isogenic or healthy control neurons (Fig. 5a; Supplementary Fig. 7, 8).We showed that these RNA foci co-localised with endogenous YY1 (Fig. 5a, b; Supplementary Fig. 7, 8) and hnRNP H (Supplementary Fig. 9) proteins.Such protein-RNA co-localisation pattern was abolished upon RNase treatment (Fig. 5a; Supplementary Figs.7-9).When compared with isogenic control neurons, the binding of endogenous YY1 protein to endogenous Fuzzy YY1 promoter regions was impaired in C9ALS/FTD neurons.YY1-mCherry overexpression in disease neurons ameliorated the defects in YY1-Fuzzy YY1 binding (Fig. 6a; Supplementary Fig. 10a).Under an overexpression condition, isogenic control neurons exogenously supplemented with YY1 also showed an enhanced YY1-Fuzzy YY1 binding (Fig. 6a).We next performed a nascent RNA capture assay 38 to determine the level of nascent Fuzzy transcript in iPSC-derived spinal motor neuron samples.When compared with the isogenic control neurons, the level of nascent Fuzzy mRNA was reduced in C9ALS/FTD neurons (Fig. 6b).Intriguingly, such reduction was rescued upon YY1-mCherry overexpression (Fig. 6b).Taken together, our data suggest that YY1 overexpression suppresses Fuzzy transcriptional dysregulation in C9ALS/FTD neurons.When transfected with a construct carrying mutant GGGGCC (pAG3-(GGGGCC) 66 ), cell death became evident in our SK-N-MC cell model, as evidenced by the release of lactate dehydrogenase 26 .We found that YY1 overexpression mitigated (GGGGCC) 66 -induced cell death (Fig. 6c).To confirm that the YY1-mediated suppression of (GGGGCC) 66 -induced cell death was not due to a reduction of mutant GGGGCC RNA expression in the co-transfected cells, we examined mutant GGGGCC expression by RT-PCR 26 and found that GGGGCC mRNA levels were not affected upon YY1 overexpression (Supplementary Fig. 10b, c).We next used a GGGGCC Drosophila model to test the modulatory function of YY1 in vivo.When expressed pan-neuronally, the expanded (GGGGCC) 36 transgene induced climbing deficits  7. n = 3 biologically independent experiments and data is presented as mean ± SEM.Source data are provided as a Source Data file.
and shortened the lifespans of the flies 39 .Overexpression of Pho (Supplementary Fig. 10d), the Drosophila orthologue of human YY1 40 , ameliorated the climbing and survival defects in (GGGGCC) 36 flies (Fig. 6d, e).The above findings confirm the modulatory role of YY1 as a suppressor in C9ALS/FTD-associated neurotoxicity.Synaptic dysfunction and degeneration have been documented as typical pathogenic features of C9ALS/FTD neurons 41,42 .Bassoon and Homer1 are key pre-and postsynaptic structural proteins, respectively, that support normal synaptic function 43 .We found that both the Bassoon and Homer1 puncta numbers along the neurites were significantly decreased in C9ALS/FTD spinal motor neurons relative to isogenic control neurons (Fig. 6f-i; Supplementary Fig. 10e, f).Our finding suggests that synaptic activity in both the pre-and postsynaptic compartments of disease neurons is potentially compromised.We found that YY1 overexpression in C9ALS/FTD iPSC-derived spinal motor neurons rescued the Bassoon and Homer1 puncta numbers, suggesting a restoration of synaptic defects in C9ALS/FTD neurons (Fig. 6f-i; Supplementary Fig. 10e, f).
Activation of the Wnt/β-catenin pathway contributes to cell death and synaptic defects in C9ALS/FTD models Fuzzy negatively regulates canonical Wnt/β-catenin activity 22,23 .We utilised a luciferase-based reporter system (TOP/FOP Flash 44 ) to examine the activity of the Wnt/β-catenin pathway in mutant GGGGCC RNA-expressing cells.Intriguingly, we found that compared with the unexpanded (GGGGCC) 2 -transfected cells, cells transfected with the pAG3-(GGGGCC) 66 construct showed stronger TOPFlash luciferase activity.This observation indicates the stimulation of the Wnt/β-catenin pathway in mutant GGGGCC RNA cells (Fig. 7a).Moreover, the mRNA levels of the Wnt/β-catenin targets Cyclin D1 (CCND1), FOS Like 1 (FOSL1) and Paired-like Homeodomain 2 (PITX2) were significantly upregulated in C9ALS/FTD neurons compared with healthy (Fig. 7b) and isogenic (Fig. 7c) control neurons.Similar changes were also detected when we analysed the transcriptomic profiles of additional C9ALS/FTD patient iPSC-derived spinal motor neuron samples from public datasets (Table 1).Consistent with transcriptomic changes, we also found that the CCND1 (Supplementary Fig. 11a), FOSL1 (Supplementary Fig. 11b) and PITX2 (Fig. 7d) protein levels were upregulated in our patient neuron model.Taken together, these data highlight the activation of the Wnt/β-catenin pathway in C9ALS/FTD neurons.
We continued to investigate the effects of CCND1, FOSL1 and PITX2 on GGGGCC-induced cell death.Of the three proteins, we found that only the knockdown of PITX2 expression attenuated (GGGGCC) 66induced cell death (Fig. 7e; Supplementary Fig. 11c-g).In contrast, PITX2 overexpression enhanced (GGGGCC) 66 -mediated cell death in SK-N-MC cells (Supplementary Fig. 11h).These results confirm the modulatory role of PITX2 in C9ALS/FTD-associated cell death.We found that knocking down Ptx1 (Supplementary Fig. 12a), the Drosophila orthologue of human PITX2 45 , in vivo rescued the climbing and survival defects in disease flies (Fig. 7f, g).Conversely, the disease phenotypes were intensified upon Ptx1 overexpression (Supplementary Fig. 12b-d).Furthermore, when PITX2 expression was knocked down, the reduction in Bassoon and Homer1 puncta numbers was ameliorated in C9ALS/FTD spinal motor neurons (Fig. 7h-k; Supplementary Fig. 12e, f).In contrast, PITX2 overexpression enhanced these synaptic deficits in C9ALS/FTD spinal motor neurons (Supplementary Fig. 13a-f).In summary, our findings illustrate the role of Wnt/β-catenin pathway activation in C9ALS/FTD pathogenesis, particularly demonstrating that the induction of PITX2 expression induces cell death and synaptic defects in C9ALS/FTD neurons.
Rescuing the dysregulation of YY1-Fuzzy-PITX2 signalling relieves synaptic defects in C9ALS/FTD neurons Next, we investigated if the restoration of YY1-Fuzzy-PITX2 signalling would ameliorate synaptic deficits in C9ALS/FTD iPSC-derived spinal motor neurons.Overexpression of BIND, but not its scrambled version BIND-S, recovered the YY1-Fuzzy YY1 promoter interactions (Fig. 8a; Supplementary Fig. 14a), followed by the restoration of Fuzzy protein expression in disease neurons (Fig. 8b).PITX2 induction was suppressed upon BIND overexpression (Fig. 8c), which also rescued the decrease in Bassoon and Homer1 puncta numbers in C9ALS/FTD spinal motor neurons (Fig. 8d-g; Supplementary Fig. 14b, c).In summary, these results suggest that restoring YY1-Fuzzy-PITX2 signalling can alleviate synaptic defects in C9ALS/FTD spinal motor neurons.

Discussion
Repeat expansion RNAs confer neurotoxicity by interacting with a range of RNA-binding proteins (RBPs), leading to the dysfunction of essential cellular pathways and, consequently, neuronal death 46 .Multiple cellular dysfunctions, such as nucleolar stress 47 , nucleocytoplasmic transport defects 48 and alternative splicing deficits 49 , have been reported to be Relative cell death (LDH assay) Fig. 6 | YY1 ameliorates the cell death and synaptic defects in C9ALS/FTD models.a The binding between YY1 and Fuzzy YY1 was disrupted in C9ALS/FTD iPSCderived spinal motor neurons, and such disruption was partially restored when YY1 was overexpressed.Overexpression of YY1 also caused the increase in YY1 and Fuzzy YY1 binding in isogenic control iPSC-derived spinal motor neurons.Both Fuzzy YY1-a and Fuzzy YY1-b DNA fragments were amplified from the endogenous Fuzzy promoter.b The level of nascent Fuzzy transcript was reduced in C9ALS/FTD iPSCderived spinal motor neurons relative to isogenic control neurons.Such reduction was mitigated upon overexpression of YY1.Overexpression of YY1 did not affect nascent Fuzzy transcript level in isogenic control neurons.YY1 overexpression caused the increase of nascent YY1 transcript in both isogenic control and disease neurons.c Overexpression of YY1 suppressed the (GGGGCC) 66 -induced cell death.Meanwhile, it did not cause a dominant toxic effect in the untransfected or (GGGGCC) 2 -expressing cells.d, e The climbing (d) and survival (e) deficits of (GGGGCC) 36 flies were alleviated when Pho was overexpressed.Overexpression of Pho did not affect the climbing ability and survival probability of (GGGGCC) 3 flies.n represents the total number of flies examined over three independent experiments.The genotypes of flies are listed in Supplementary Table 6.f-i Bassoon (f) and Homer1 (h) puncta numbers were reduced in C9ALS/FTD iPSC-derived spinal motor neurons.Such reduction was alleviated upon overexpression of YY1.The number of Bassoon (f) and Homer1 (h) puncta was not affected in isogenic control iPSC-derived spinal motor neurons when YY1 was overexpressed.Scale bars: 20 μm.g and i are the quantifications of f and h, respectively.n represents the total number of neurites examined over three independent experiments.One-way ANOVA followed by post hoc Tukey's test was used in panels (a-d), (g) and (i).Log-rank (Mantel-Cox) test was used in (e).The exact P values are listed in Supplementary Table 7. n = 3 biologically independent experiments and data is presented as mean ± SEM.Source data are provided as a Source Data file.
conferred by mutant GGGGCC RNA expression.In this study, we found that GGGGCC RNA interacts with the transcriptional regulator YY1 (Fig. 3a).Our data showed that such RNA-protein interaction impairs the binding of YY1 to the Fuzzy promoter (Fig. 2f), downregulates Fuzzy transcription (Fig. 2g) and activates the canonical Wnt/β-catenin pathway (Fig. 7a).The induction of the Wnt/β-catenin pathway target gene PITX2 triggers cell death and synaptic defects in C9ALS/FTD models (Fig. 7).Collectively, our study uncovered the interplay among GGGGCC RNA, YY1 and Fuzzy promoter DNA in C9ALS/FTD neurons.We showed that GGGGCC RNA prevents YY1 from binding to the Fuzzy promoter, thereby inhibiting Fuzzy transcription, which consequently stimulates the canonical Wnt/β-catenin pathway and leads to C9ALS/FTD-associated neurodegeneration (Fig. 9).Emerging evidence emphasises the pathogenic roles of developmental pathways, such as the Notch, Hedgehog and Wnt/β-catenin pathways, in human neurological disorders 50 .The Wnt/β-catenin pathway has previously been studied in the context of ALS 51 .In postmortem spinal cord samples obtained from patients with sporadic The transcript levels of CCND1, FOSL1, and PITX2, but not other Wnt-responsive genes, were significantly upregulated in C9ALS/FTD patient iPSC-derived spinal motor neurons when compared to the healthy (b) and isogenic (c) control neurons.d The protein level of PITX2 was upregulated in C9ALS/FTD iPSC-derived spinal motor neurons compared to the healthy and isogenic control neurons.e The (GGGGCC) 66induced cell death was suppressed upon knockdown of PITX2.No dominant cytotoxic effect was detected in the untransfected or (GGGGCC) 2 -expressing cells when PITX2 was knocked down.f, g Knockdown of Ptx1 ameliorated the climbing (f) and survival (g) deficits of (GGGGCC) 36 flies, and it did not affect the climbing ability and survival probability of (GGGGCC) 3 flies.n represents the total number of flies examined over three independent experiments.The genotypes of flies are listed in Supplementary Table 6.h-k Knockdown of PITX2 rescued the reduction of Bassoon (h) and Homer1 (j) puncta numbers in C9ALS/FTD iPSC-derived spinal motor neurons.The number of Bassoon (h) and Homer1 (j) puncta was not affected in isogenic control iPSC-derived spinal motor neurons when PITX2 was knocked down.Scale bars: 20 μm.i and k are the quantifications of h and j, respectively.n represents the total number of neurites examined over three independent experiments.Two-tailed unpaired Student's t-test was used in panels (a-c).Oneway ANOVA followed by post hoc Tukey's test was used in panels (d-f), (i) and (k).One-way ANOVA followed by post hoc Dunnett's test was used for the comparison between disease and isogenic control neurons in (d).Log-rank (Mantel-Cox) test was used in panel (g).The exact P values are listed in Supplementary Table 7. n = 3 biologically independent experiments and data is presented as mean ± SEM.The illustration in panel 7a was created using Adobe Illustrator.Source data are provided as a Source Data file.
ALS, the expression of several Wnt ligands and receptors was found to be elevated 52 .Similar findings were reported in SOD1 G93A ALS transgenic mouse models 53 , the spinal cord samples of which showed extensive nuclear accumulation of β-catenin and induction of the target gene CCND1 54 .In addition to ALS, Wnt signalling has been implicated in FTD pathogenesis.Using a progranulin (GRN)-silenced human FTD neural stem cell model, Rosen et al. 55 reported that the transcription of Wnt signalling components was broadly dysregulated in GRN-inactivated cells.Similar findings have been reported by another group using FTD patient-derived iPSCs carrying a GRN haploinsufficiency mutation 56 .They also demonstrated that genetic correction of the mutation or treatment with a Wnt signalling inhibitor mitigated the corticogenesis defects in the FTD iPSCs 56 .These findings underscore the tight associations between Wnt signalling, ALS and FTD pathogenesis.In C9ALS/FTD iPSC-derived spinal motor neurons, we observed the induction of several Wnt/β-catenin targets, namely, CCND1, FOSL1 and PITX2, at both the mRNA and protein levels (Fig. 7b-d; Supplementary Fig. 11a, b).Our findings describe Wnt/βcatenin pathway activation in C9ALS/FTD neurons.
We previously reported that excess Fuzzy protein expression is detrimental to neuronal health 8 .Fuzzy promotes the aggregation of Dishevelled (Dvl), which recruits the small GTPase Rac1 57 to activate downstream MAPK-caspase signalling and trigger apoptosis 8 .In Fuzzy −/− cells, Dvl aggregation has been shown to be inhibited; this is accompanied by an increase in evenly distributed Dvl 8 , a protein conformation that promotes the activation of the Wnt/β-catenin pathway 58 .The transcription of Wnt/β-catenin target genes is known to be induced in Fuzzy −/− mouse embryos 23 , suggesting a negative regulatory effect of Fuzzy on Wnt/β-catenin pathway activity.Here, we showed that Fuzzy expression was downregulated in C9ALS/FTD spinal motor neurons (Fig. 1b) and that Wnt/β-catenin pathway targets were upregulated in disease neurons (Fig. 7b, c).In particular, the upregulation of PITX2 expression intensified cell death and synaptic defects in C9ALS/FTD models (Supplementary Figs.11h, 13a-f).Taken together, these findings suggest that tight control of Fuzzy protein expression is crucial for the maintenance of neuronal viability.YY1 can function both as an activator and a repressor of gene transcription 32 .In this study, we demonstrated that YY1 modulates Fuzzy transcription through binding to two independent Fuzzy YY1 sites in the Fuzzy promoter.Our findings illustrate that YY1 plays transcriptional activator and repressor roles at Fuzzy YY1-a and Fuzzy YY1-b , respectively (Fig. 2c).Interestingly, when YY1-Fuzzy YY1-a interaction was impaired, the Fuzzy promoter activity was dramatically reduced (~50fold; Fig. 2c).In contrast, only a mild induction effect (~2.5-fold) was detected when YY1-Fuzzy YY1-b interaction was impaired (Fig. 2c).This difference could be due to the fact that YY1 utilises ZF3 to bind to Fuzzy YY1-a , but ZF4 to bind to Fuzzy YY1-b (Fig. 2b).Indeed, it was previously reported that mutating ZF3 resulted in a total disruption of YY1 activity, while no such effect was observed in ZF4 mutant 30 .This highlights that ZF3 plays a more critical role than ZF4 in regulating YY1 transcriptional activity.
In addition to the transcription of GGGGCC RNA from the sense strand of the pathogenic allele, the transcription of CCCCGG RNA from the antisense strand also occurs in C9ALS/FTD neurons 59 .Similar to GGGGCC RNA, the CCCCGG RNA possesses protein-binding capability towards key cellular factors such as a subset of heterogeneous nuclear ribonucleoproteins (hnRNPs) 60,61 .Additional RBPs, such as nucleolin (NCL) and DEAD-box helicase 3 X-linked, show specificity in binding to GGGGCC RNA 47,62 .In contrast, hnRNP K, a cytosine-rich RNA-binding protein, preferentially interacts with CCCCGG RNA 47 .It has been reported that GGGGCC RNA adopts G-quadruplex (G4) conformations 63,64 , whereas CCCCGG RNA forms a double helix with tandem C:C mismatches 65 .In this study, we found that YY1 co-localised with GGGGCC RNA foci, but not with CCCCGG RNA foci, in C9ALS/FTD spinal motor neurons (Fig. 5a; Supplementary Fig. 15).Like YY1, NCL is a G4-binding protein 66 .We previously showed that BIND, a 21-amino acid peptide derived from the RNA recognition motif of NCL 67 , can bind to GGGGCC RNA 26 .We found that BIND overexpression almost fully reversed Fuzzy downregulation and PITX2 induction in C9ALS/FTD spinal motor neurons (Fig. 8b, c).This finding further validates the role of GGGGCC RNA as the driver of downstream pathogenic YY1-Fuzzy-PITX2 signalling to induce neuronal dysfunction in C9ALS/FTD.In conclusion, our study revealed a pathogenic signalling cascade initiated by GGGGCC RNA that involves YY1-Fuzzy transcriptional dysregulation and aberrant Wnt/β-catenin pathway induction.This cascade leads to C9ALS/FTD-associated synaptic defects and cell death.An in-depth understanding of this pathogenic signalling axis could shed light on the disease mechanisms underlying C9ALS/FTD and provide new therapeutic targets to suppress C9ALS/FTD pathogenesis.

Ethics statement
Biological and chemical safety approval (14122815) from The Chinese University of Hong Kong was obtained for this study.All animal procedures were approved by the CUHK Animal Experimentation Ethics Committee (and their care was in accordance with the institutional guidelines).All iPSC lines were derived from human skin biopsy fibroblasts, collected under ethical approval granted by the South Wales Research Ethics Committee (WA/12/0186) in the Martin Stem Cell Facility, University of Oxford, under standardized protocols.

Click-iT nascent RNA capture assay
The Click-iT nascent RNA capture assay (C10365, Thermo Fisher Scientific) was performed following the manufacturer's instructions.The disease and control iPSC-derived spinal motor neurons differentiated for 30 days were labelled with 0.5 mM 5-ethynyl uridine (EU) at 37 °C with 5% CO 2 for 1 h.The total RNA was subsequently harvested using RNeasy Mini Kit (74104, Qiagen) supplemented with DNase treatment (79254, Qiagen).The biotin-azide was conjugated to EU to generate the biotinylated RNA.For each sample, 12 μL of Streptavidin magnetic beads were mixed with 200 ng biotinylated RNA to capture EUlabelled nascent transcripts.The on-beads cDNA synthesis was performed using SuperScript VILO TM cDNA synthesis kit (11754050, Thermo Fisher Scientific), followed by the qPCR to determine levels of nascent Fuzzy and YY1 transcripts using primers listed in Supplementary Table 5.

Fluorescence recovery after photobleaching (FRAP)
The SK-N-MC cells were seeded onto a 35-mm coverglass bottom confocal dish (SPL-100350, SPL Life Sciences Co., Ltd.).Twenty-four hours later, the cells were transfected with 1 μg of pAG3-(GGGGCC) 2/66 -MS2, 0.5 μg of pMS2CP-YFP, together with 0.2 μg of phnRNP H-mCherry or 0.5 μg of pYY1-mCherry construct for 48 hours.In the FRAP experiment, cells were maintained in DMEM without phenol red (31053028, Thermo Fisher Scientific).Region of interest (ROI) was selected and photobleached using the FRAP module on a TCS SP8 high speed imaging system (Leica Microsystems).All pre-bleach, photobleaching, and post-bleach steps were controlled automatically by the "Zoom in" mode of Leica Application Suite X software.Cells were imaged before bleach (pre-bleach), followed by the bleach with 2 times 100% power of the 561-nm laser, and post-bleach for twenty iterations.Only representative images are shown.The fluorescent intensity of the bleached ROI at each timepoint was automatically calculated on the Leica Application Suite X software.These fluorescent intensities were normalized against the fluorescent intensity from the pre-bleach ROI to determine the fluorescent recovery.
The membrane was washed three times with 1× TBST each for 10 min before being subjected to the incubation of secondary antibodies at 25 °C for 1 h.Secondary antibodies used were HRPconjugated goat anti-rabbit IgG (H + L) (11-035-045, 1:5000) and HRPconjugated goat anti-mouse IgG (H + L) (115-035-062, 1:10,000) from Jackson ImmunoResearch (West Grove, USA).The membrane was washed three times with 1× TBST each for 10 min, followed by chemiluminescent signal detection.The signal was developed using Immobilon Forte Western HRP substrate (WBLUF0100, Merck Millipore), and the images were captured and processed using ChemiDoc TM Touch Imaging System.β-tubulin was used as the loading control.Only representative blots are shown.

Lactate dehydrogenase (LDH) cytotoxicity assay
The LDH cytotoxicity assay was performed using a CytoTox 96® nonradioactive cytotoxicity assay kit (G1780, Promega) according to the manufacturer's instructions.Briefly, SK-N-MC cells were cultured in 24well plates, after transfection, the cells were applied for the cytotoxicity assay.Both the absorbance of LDH enzyme released into the culture medium (Absorbance supernatant ) and from attached cells (Absorbance attached ) were obtained on a Spark® multimode microplate reader (Tecan, Switzerland).The relative cell death was calculated using the following equation and the data from experimental groups were normalized to respective controls.

Relative cell death =
Absorbance supernatant Absorbance supernatant + Absorbance attached

Electrophoretic mobility shift assay
Both DNA and RNA oligonucleotides (Supplementary Table 4) were synthesized by Integrated DNA Technologies and Dharmacon and dissolved in buffer containing 8 mM Na 2 HPO 4 , 185 mM NaCl and 1 mM EDTA.The DNA and RNA oligonucleotides were denatured at 60 °C for 8 min, refolded at 37 °C for 30 min, and then gradually cooled down to 25 °C prior to use.The (GGGGCC) 8 RNA and randomised GC-rich RNA were denatured at 95 °C for 5 min and then gradually cooled down to 25 °C prior to use.The recombinant His-YY1 full-length , His-YY1 ZF1+ZF2+ZF3 and His-YY1 ZF1+ZF2 proteins were produced by GenScript and dissolved in buffer containing 50 mM Tris-HCl, 500 mM NaCl, 10% Glycerol, pH 8.0.The denatured DNA or RNA oligonucleotide (2.5 nM of each) was mixed with different concentrations of recombinant YY1 proteins and 20 U RNaseOUT recombinant ribonuclease inhibitor (10777019, Thermo Fisher Scientific) in binding buffer (10 mM HEPES-KOH, 12 mM Tris-HCl, 50 mM NaCl, 50 mM KCI, 5 mM MgCl 2 , 100 μM ZnCl 2 , 0.01% NP-40, 5% glycerol, 2.5 mM DTT, pH 7.4).The mixtures were incubated at 25 °C in dark for 30 min before being loaded onto 6% nondenaturing polyacrylamide gels for electrophoresis on ice in 0.5x TB running buffer (45 mM boric acid, 45 mM Tris, 50 μM ZnCl 2 ).
For the competition assays, 2.5 nM of Cy5-Fuzzy YY1-a or Cy5-Fuzzy YY1-b DNA was mixed with 2.65 mM recombinant His-YY1 full-length protein (ab187479, Abcam) and 20 U RNaseOUT recombinant ribonuclease inhibitor in binding buffer.An increasing amount of (GGGGCC) 8 RNA (5′-GGGGCCGGGGCCGGGGCCGGGGCCGGGGCCGGGGCCGGGGCCGG GGCC-3′) was introduced to the YY1-Fuzzy YY1 DNA binding reactions.The mixtures were incubated at 25 °C in dark for 30 min and then loaded onto 6% nondenaturing polyacrylamide gels for electrophoresis on ice in 0.5x TB running buffer.The gels were visualized, and images were captured using ChemiDoc TM MP System (17001402, Bio-Rad).Only representative gels are shown.The band intensities of both free DNA/ RNA probes (Intensity free probe ) and YY1-nucleotide complexes that are resolved in the gel (Intensity complex ) were quantified using the ImageJ software.The percentage of shifted DNA/RNA signals was calculated by the following equation: DNA/RNA fraction bound (%) = Intensity complex / (Intensity free probe + Intensity complex ) × 100%.The obtained values were used to plot the YY1-RNA binding curves.The binding affinity (K d ) was determined using the non-linear regression model on the GraphPad Prism software (version 9.2.0).

Fluorescence polarisation (FP) assay
The FAM-labelled (GGGGCC) 8 RNA oligonucleotide (Supplementary Table 4) was dissolved in buffer containing 8 mM Na 2 HPO 4 , 185 mM NaCl and 1 mM EDTA.The RNA oligonucleotide was denatured at 60 °C for 8 min, refolded at 37 °C for 30 min, and then gradually cooled down to 25 °C prior to use.The denatured RNA oligonucleotide (2.5 nM) was mixed with different concentrations of recombinant His-YY1 full-length protein in binding buffer (50 mM Tris-HCl, 100 mM KCl, 2.5 mM MgCl 2 , 100 μM ZnCl 2 , pH 7.4).The mixture was incubated at 25 °C in dark for 30 min.The fluorescence polarisation was measured on a Spark® multimode microplate reader with the excitation and emission wavelengths of 485 nm and 535 nm, respectively.The parallel (I parallel ) and perpendicular (I perpendicular ) fluorescence intensities were determined by the SparkControl software (version 2.1), following by the calculation of millipolarisation (mP) units = 1000 × (I parallel − I perpendicular ) / (I parallel + I perpendicular ).The binding affinity (K d ) was determined using the non-linear regression model on the GraphPad Prism software (version 9.2.0).

Drosophila climbing and survival assays
The 2 days post eclosion (dpe) flies were transferred to fly food containing 200 μM mifepristone (RU486, ab120356, Abcam).The RU486 was used to induce transgene expression.The climbing ability of flies was measured by negative geotaxis assay.Flies from each experimental group were anesthetized and placed in a vertical plastic column.After an hour of recovery in 25 °C incubator, flies were banged to the bottom, and the time they used to climb across 6 cm in 30 s was recorded.For those who failed to climb across 6 cm in 30 s, the climbing time was marked as 30 s.The ages of flies used are: 14 dpe (Figs.6d and 7f) and 10 dpe (Supplementary Fig. 12c).For the survival assay, the survival rate of flies was recorded daily until flies from each experimental group were all dead.The survival plots were generated by using the GraphPad Prism software (version 9.2.0).

Analysis of public RNA-seq datasets
The raw sequencing data from public RNA-seq datasets were downloaded from Gene Expression Omnibus (GEO) with the accession codes GSE143743 and GSE168831.The datasets were imported to Partek Flow Genomic Analysis Software for analysis.The reads were aligned to the hg38 build of the human genome under the "STAR" mode.The aligned reads were further quantified to Ensembl Transcripts release 105 annotation model to generate gene counts.After filtering out genes with low read counts, the filtered counts among different samples were normalized using the "Median ratio" method.The DESeq2 method was subsequently used for the analysis of normalized counts to obtain differentially expressed genes.The lists of differentially expressed genes from NeuroLINCS dataset and AnswerALS data portal were downloaded directly from the published paper 72 .

Software
The intensity of the protein or DNA bands were quantified using the ImageJ software (https://imagej.nih.gov/ij/) 73.The differential expressed gene lists were generated using the Partek Flow Genomic Analysis Software (https://www.partek.com/partek-flow/).The illustrations were created using Adobe Illustrator 27.0.1 and BioRender.com.Assembly of panels in finalized figures was done using Adobe Illustrator 27.0.1.

Statistical analysis
The two-tailed, unpaired Student's t test or Mann-Whitney U test was used for the comparison between two experimental groups.One-way ANOVA followed by post hoc Tukey's test or one-way ANOVA followed by post hoc Dunnett's test was applied when comparing three or more groups.Log-rank (Mantel-Cox) test was used for survival analysis.*, **, ***, and **** represent P < 0.05, P < 0.01, P < 0.001, and P < 0.0001, respectively, which are considered statistically significant.ns indicates no significant difference.The violin plots show the median (dashed lines), and 25th and 75th quartiles (dotted lines).GraphPad Prism version 9.2.0 was used to plot data and perform statistical analysis.

Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Fig. 1 |
Fig.1| Expression of mutant GGGGCC RNA causes downregulation of Fuzzy promoter activity.a, b The Fuzzy/Fuzzy transcript (a) and protein (b) levels were downregulated in C9ALS/FTD patient iPSC-derived spinal motor neurons when compared to the healthy and isogenic control neurons.c Illustration of the Fuzzy promoter luciferase reporter and different GGGGCC expression constructs used in this study.d Overexpression of pAG3-(GGGGCC) 66 construct suppressed Fuzzy promoter luciferase activities at Fuzzy -2732/+574 , Fuzzy -2032/+574 and Fuzzy -1332/+574 regions, whereas the activity of Fuzzy -632/+574 luciferase construct was not altered.The (GGGGCC) 66 -responsive region, Fuzzy -1332/-633 , is in yellow.e Fuzzy promoter activity was downregulated in (GGGGCC) 106 -RO-expressing cells, while such downregulation was restored upon treatment of BIND peptide.f The (GGGGCC)66 -and (GGGGCC) 106 -RO-responsive element was further narrowed down to Fuzzy -1332/-1143 region, which is in red.g Schematic representation of the locations and sequences of Fuzzy YY1-a (in red font) and Fuzzy YY1-b (in blue font) sites in Fuzzy -1332/+574 promoter

Fig. 2 |
Fig.2| GGGGCC RNA perturbs the binding of YY1 to Fuzzy promoter, leading to Fuzzy downregulation.a The binding of recombinant YY1 protein to Fuzzy YY1-a DNA is stronger than to Fuzzy YY1-b DNA.The R 2 of the YY1-Fuzzy YY1-a and YY1-Fuzzy YY1-b binding curves are 0.9915 and 0.9818, respectively.The Fuzzy promoter region that carries one copy of Fuzzy YY1-a or Fuzzy YY1-b site was synthesized and used in the EMSA.The sequences of the Cy5-labelled Fuzzy YY1-a DNA and Fuzzy YY1-b DNA (each of 20 base pairs in length) probes are listed in Supplementary Table4.b The ZF3 in YY1 is required for interacting with Fuzzy YY1-a , while ZF4 mediates the interaction between YY1 and Fuzzy YY1-b .Both Fuzzy YY1-a and Fuzzy YY1-b DNAs were amplified from the endogenous Fuzzy gene promoter.c Mutation of Fuzzy YY1-a led to downregulation of Fuzzy promoter activity, whereas mutation of Fuzzy YY1-b caused the opposite upregulation effect.d Knockdown of YY1 led to the inhibition of Fuzzy promoter activity.e Addition of GGGGCC RNA perturbed the formation of protein-DNA complexes between recombinant YY1 protein and Fuzzy YY1-a /Fuzzy YY1-b DNA, leading to the

Fig. 3 |
Fig. 3 | The GGGGCC RNA binds to YY1 protein and recruits it to RNA foci.a The binding between recombinant His-YY1 full-length protein and (GGGGCC) 8 RNA was detected by EMSA.The R 2 of the YY1-(GGGGCC) 8 RNA binding curve is 0.9914.The sequence of the Cy5-labelled (GGGGCC) 8 RNA (48 bases in length) probe is listed in Supplementary Table 4. b The fluorescence polarisation assay was performed to demonstrate the binding between recombinant His-YY1 full-length protein and (GGGGCC) 8 RNA.The R 2 of the YY1-(GGGGCC) 8 RNA binding curve is 0.9881.The sequence of the FAM-labelled (GGGGCC) 8 RNA (50 bases in length) probe is listed in Supplementary Table4.c The endogenous hnRNP H protein (green) was found colocalised with GGGGCC RNA foci (red) in (GGGGCC) 66 -expressing SK-N-MC cells.Scale bars: 5 μm.No GGGGCC RNA foci were detected in (GGGGCC) 2 -expressing cells or cells that received RNase treatment.d, e The co-localisation between endogenous YY1 protein (green) and GGGGCC RNA foci (red) was detected in (GGGGCC) 66 -transfected SK-N-MC cells by two anti-YY1 antibodies raised against different epitopes.Scale bars: 5 μm.No GGGGCC RNA foci were detected in (GGGGCC) 2 -transfected cells or cells that received RNase treatment.f is the quantification of (c-e).The number of GGGGCC foci-positive cells counted over three independent experiments were: hnRNP H: 180; YY1 (Abcam): 299; YY1 (Proteintech): 182.g, h The formation of GGGGCC RNA foci was detected when pAG3-(GGGGCC) 66 -MS2, but not pAG3-(GGGGCC) 2 -MS2, was co-transfected with MS2CP-YFP in SK-N-MC cells.The recovery of YY1-mCherry (g) and hnRNP H-mCherry (h) fluorescent signals after photobleaching was much less effective in GGGGCC foci formation cells.The circled regions indicate the region of interest (ROI) that underwent photobleaching.The pre-bleach and post-bleach fluorescent signals in the ROI were recorded.n represents the total number of cells examined over three independent experiments.Two-tailed unpaired Mann-Whitney U test was used in panels (g) and (h).The exact P values are listed in Supplementary Table7.n = 3 biologically independent experiments and data is presented as mean ± SEM.Source data are provided as a Source Data file.

Fig. 5 |
Fig. 5 | The YY1 protein is recruited to GGGGCC RNA foci in C9ALS/FTD iPSCderived spinal motor neurons.a The endogenous YY1 protein (green) was recruited to the GGGGCC RNA foci (red) in C9ALS/FTD iPSC-derived spinal motor neurons.No such recruitment was detected in isogenic control spinal motor neurons or neurons that received RNase treatment.b is the quantification of Fig. 5a and Supplementary Fig. 8.The number of GGGGCC foci-positive neurons counted over three independent experiments were: C901-06: 127; C901-07: 103; C902-02: 122; C902-03: 152; C904-01: 176; C904-12: 143.Data are presented as mean ± SEM.No GGGGCC foci formation was detected in isogenic control iPSC-derived spinal motor neurons or neurons treated with RNase.Arrows indicate the co-localisation between the endogenous YY1 protein and GGGGCC RNA foci.The cell nuclei were stained with Hoechst 33342 (blue).Scale bars: 2 μm.Source data are provided as a Source Data file.

Fig. 7 |
Fig. 7 | Induction of Wnt/β-catenin target gene PITX2 contributes to cell death and synaptic defects in C9ALS/FTD models.a When compared to the unexpanded (GGGGCC) 2 -expressing cells, overexpression of pAG3-(GGGGCC) 66 in SK-N-MC cells stimulated the TOP flash, but not FOP flash luciferase activity.b, cThe transcript levels of CCND1, FOSL1, and PITX2, but not other Wnt-responsive genes, were significantly upregulated in C9ALS/FTD patient iPSC-derived spinal motor neurons when compared to the healthy (b) and isogenic (c) control neurons.d The protein level of PITX2 was upregulated in C9ALS/FTD iPSC-derived spinal motor neurons compared to the healthy and isogenic control neurons.e The (GGGGCC) 66induced cell death was suppressed upon knockdown of PITX2.No dominant cytotoxic effect was detected in the untransfected or (GGGGCC) 2 -expressing cells when PITX2 was knocked down.f, g Knockdown of Ptx1 ameliorated the climbing (f) and survival (g) deficits of (GGGGCC) 36 flies, and it did not affect the climbing ability and survival probability of (GGGGCC) 3 flies.n represents the total number of flies examined over three independent experiments.The genotypes of flies are listed in

Fig. 8 |
Fig.8| Overexpression of BIND rescues dysregulation of YY1-Fuzzy-PITX2 signalling axis and suppresses synaptic defects in C9ALS/ FTD spinal motor neurons.a Overexpression of BIND rescued the defects in binding between YY1 and both Fuzzy YY1-a and Fuzzy YY1-b promoter sites.Overexpression of BIND-S failed to elicit a similar rescuing effect.Both Fuzzy YY1-a and Fuzzy YY1-b DNA fragments were amplified from the endogenous Fuzzy promoter.b, c Overexpression of BIND, but not BIND-S, restored the Fuzzy reduction (b) and PITX2 induction (c) in C9ALS/FTD spinal motor neurons.d-g Overexpression of BIND protein suppressed the reduction of Bassoon (e) and Homer1 (g) puncta numbers in C9ALS/FTD iPSC-derived spinal motor neurons, whereas BIND-S overexpression did not cause a similar suppression effect.Overexpression of neither BIND nor BIND-S affects the Bassoon (e) and Homer1 (g) puncta numbers in the isogenic control iPSC-derived spinal motor neurons.Scale bars: 20 μm. e and g are the quantifications of d and f, respectively.n represents the total number of neurites examined over three independent experiments.Two-tailed unpaired Student's t-test was used in panel (a).One-way ANOVA followed by post hoc Tukey's test was used in panels (b), (c), (e) and (g).The exact P values are listed in Supplementary Table7.n = 3 biologically independent experiments and data is presented as mean ± SEM.Source data are provided as a Source Data file.

Fig. 9 |
Fig.9| Graphical summary of the current study.In normal spinal motor neurons, YY1 binds to Fuzzy promoter at Fuzzy YY1-a and Fuzzy YY1-b sites to control the normal level of Fuzzy protein.The canonical Wnt/β-catenin pathway is not activated, and no neurodegeneration occurs.In C9ALS/FTD spinal motor neurons, YY1 binds to GGGGCC RNA.The binding of YY1 to the Fuzzy promoter is impaired, followed by the downregulation of the Fuzzy protein level.The Wnt/β-catenin pathway is activated.The induction of PITX2 proteins consequently contributes to the degeneration of disease neurons.The illustration was created using BioRender.com.

Table 1 |
Summary of the analysis of CCND1, FOSL1 and PITX2 levels from public transcriptomic datasets